Chromatographic techniques are important tools for the identification and separation of complex samples. The basic principle underlying chromatographic techniques is the separation of a mixture into individual components by transporting the mixture in a moving fluid through a retentive media. The moving fluid is typically referred to as the mobile phase and the retentive media is typically referred to as the stationary phase. The separation of the various constituents of the mixture is based on differential partitioning between the mobile and stationary phases, and subtle differences in a component's partition coefficient resulting in differential retention on the stationary phase, thus changing the separation.
There are a number of different types of chromatographic techniques, each having different operating conditions and providing different advantages for separating and analyzing samples. For example, some known techniques include liquid chromatography (LC), high-performance liquid chromatography (HPLC), ultra-high performance liquid chromatography UHPLC), gas chromatography (GC), and supercritical fluid chromatography (SFC). One difference between these techniques is the type of mobile phase material used. For example, LC and HPLC use liquids, whereas GC uses a gas as the mobile phase. SFC uses a supercritical fluid as the mobile phase for sample separation.
The supercritical fluid (SF) phase of matter is defined by the supercritical point, that is, the temperature and pressure values at which liquid and vapor have the same density and the fluid cannot be liquefied by increasing the pressure. The unique physical properties of supercritical fluids make them attractive mobile phase fluids for use in supercritical fluid chromatography (SFC). For example, supercritical fluids (or near supercritical fluids) offer the ability to dissolve samples readily like a liquid (e.g. in high-performance liquid chromatography, “HPLC”), and carry them through a stationary phase like a gas (e.g. in gas chromatography, “GC”).
Another important property of SFs is that SFs provide high resolution chromatography at much lower temperatures than those used for GC, thereby enabling analysis of proteins and other biopolymers that can be sensitive to heat. For example, an analyte dissolved in supercritical CO2 can be recovered by reducing the pressure and allowing the sample to evaporate under ambient laboratory conditions.
Because SFC typically uses CO2 as a primary component of the mobile phase (e.g. CO2 is about 100% of the mobile phase, CO2 is about 99% of the mobile phase, CO2 is about 97% of the mobile phase, CO2 is about 95% of the mobile phase, CO2 is about 90% of the mobile phase, CO2 is about 80% of the mobile phase, etc.), SFC separations are inexpensive, innocuous, eco-friendly, and non-toxic. There is typically no need for the use of volatile solvent(s) (e.g., hexane). Finally, SFs, such as CO2, have higher diffusion constants and lower viscosities relative to liquid solvents.
Supercritical fluids are considered compressible fluids. Unlike incompressible fluids such as liquids used for HPLC (e.g. water, methanol, hexanes, etc.) which instantly or nearly instantly equilibrate pressure evenly throughout the system when the local pressure changes at one point, compressible fluids such as supercritical fluids can experience temporary heterogeneity overall in response to a local pressure change. In the context of SFC, this means that as the supercritical fluid (or a near supercritical fluid) moves down the length of a chromatography column or separating segment, it expands. Correspondingly, as the supercritical mobile phase expands, it also cools. This phenomenon has been elucidated by A. Tarafder, G. Guiochon, J. Chromatogr. A, 1218 (2011) 7189; D. P. Poe, J. Chromatogr. A, 785 (1997), 129; D. P. Poe, J. J. Schroden, J. Chromatogr. A, 1216 (2009), 7915; K. Kaczmarski, D. P. Poe, G. Guiochon, J. Chromatogr. A, 1217 (2010) 6578. (The foregoing publications are hereby incorporated by reference in their entirety).
In some embodiments, carbon dioxide (CO2) is used as the mobile phase or as a primary component of the mobile phase (i.e., CO2-based chromatography). In some embodiments, chromatography using CO2 as the mobile phase can be carried out at, near or below the supercritical point (e.g. as a liquid or a gas). When pumped as a liquid or a gas, CO2 has a variable density due to temperature and pressure changes; that is, the density of CO2 in the liquid or gas phase can vary dramatically as compared to other LC or HPLC mobile phases such as water, acetonitrile, methanol, or hexanes over small temperature or pressure changes. In general, CO2 when used as a mobile phase in chromatography is considered to be a compressible fluid.
The cooling of the supercritical mobile phase or carbon dioxide mobile phase as it passes through a chromatography column (e.g. separating segment) leads to consequences for the resulting separation. One issue is that the cooling can be enough to lead to a change in state of the mobile phase. For instance, excessive cooling might cause a supercritical or near supercritical fluid to lose its supercritical or near supercritical properties and become a liquid. This would negate many of the advantages of SFC highlighted above. For example, a change of state such as becoming a liquid might increase the mobile phase viscosity and decrease the diffusion coefficients. This can lead to lower efficiency separations and higher column pressure drops. Another difficulty is that as the mobile phase cools locally, it becomes heterogeneous. For example, in addition to experiencing a temperature gradient, the mobile phase can demonstrate different physical properties such as density and viscosity along the length and radius of the separating column. Such heterogeneity of the mobile phase leads to distorted peak shape, as well as less efficient separations. These and other considerations make SFC and/or CO2-based chromatography less effective due to unplanned changes of state or changes in density, especially from the impact of cooling.